The present invention provides novel compounds that target nucleic acids to the cell nucleus. Further, this invention generally relates to the transformation of cells, particularly mammalian cells, with exogenous DNA or other nucleic acids. More particular, the invention relates to a method for introducing nucleic acids into the nucleus of cells with the help of such compounds. In addition pharmaceutical preparations containing such compounds and the use of such compounds for gene therapy are also in the field to which the invention relates.
All references cited herein in short form are outlined in detail in the list of references. The use of gene therapy to treat diseases of both genetic and infectious origin has increasingly become the focus of biomedical research. Accordingly, there have been numerous attempts to develop appropriate delivery systems, based on either recombinant viruses or non-viral vectors.
Several methods have been developed for introducing exogenous DNA molecules into eukaryotic cells for the production of transiently-and stably transfected cells. These methods include physical and chemical systems such as electroporation, microinjection, dextran, liposomes, calcium phosphate or polyethylenimine (PEI) mediated DNA uptake or cell fusion, and microprojectile bombardment. In addition, viral vectors have been used for DNA delivery into cells.
Although physical and chemical methods relatively efficiently overcome the plasma membrane of the cell, it is still unclear how DNA introduced into the cell by these methods penetrate the nuclear envelope. One of the current hypothesis is that the exogenous DNA that survives cytoplasmic degradation is incorporated into the nascent nucleus during cell division. Thus, cytoplasmic degradation as well as the capability of a transfected cell to divide will limit the efficacy of DNA uptake into the nucleus. Furthermore, quiescent, non-dividing cells are rarely transformed by these methods. However, targeting of quiescent cells is of primary importance for somatic gene therapy since a large proportion of somatic cells are non-dividing.
Another limitation of conventional physical and chemical methods is that they cannot provide specificity for particular cell types, i.e. by using a receptor mediated uptake approach. However, this is a highly desired goal if particular cells are to be targeted in tissues or intact organisms, as e.g. in gene therapy applications.
In an attempt to overcome some of the above drawbacks recombinant viral vectors are used for cell transformation. For example, viral systems derived from Adenovirus, Adeno-associated virus, Herpes simplex virus and HIV are being evaluated for targeting of quiescent cells. However, such viral systems pose various problems that are well-known in the art regarding safety in production and application, production costs, efficiency of transfection, duration of expression and amount of DNA that can be packaged, depending on the particular approach used. For example, the use of adenoviral systems is limited by the induction of immune responses to viral antigens with subsequent clearance of transducted cells, thereby strongly diminishing the prospects for long term gene expression. A major safety issue when viral vectors are used is i.e. the generation of replication competent particles during the in vivo packaging of recombinant viruses. This problem is absent if non-viral gene transfer systems are used.
A major advantage of some viral gene delivery systems as compared to conventional physical and chemical methods is the ability of viral vectors to target their DNA-load to the nucleus of the transduced cell, thereby increasing transformation efficiency.
An approach to target DNA into the cell nucleus would be to make use of the cell""s own transport mechanisms that specifically guide cytoplasmic molecules through the nuclear pore into the nucleus. In example, certain transcription factors upon activation specifically translocate into the nucleus and, thus, such transcription factors could be used to target molecules to the nucleus. Steroid hormone receptors are an example of transcription factors located in the cytoplasm. They are activated by the binding of steroid hormones and subsequently localize to the nucleus. Use of these receptors for gene delivery systems could therefore accomplish nuclear targeting of the transfected DNA. Via nuclear targeting of the transfected DNA cells could be transfected more efficiently, because breakdown of the nuclear envelope during cell division would not be required to incorporate the transfected DNA. In particular, non-dividing cells could be transformed more efficiently. In addition, cellular targeting the DNA exclusively to cells that express the particular receptor used could be accomplished.
Petros et al. (WO 96/03875) describe a gene delivery system for nucleic acids to cells that comprises a steroid moiety capable of binding to an androgen receptor, wherein the steroid moiety is covalently linked lo a cationic salt, i.e. poly-L-lysine via an ester, an amide or a disulfide bond.
Efficient transformation of cells with this approach is largely dependent on the intracellular stability of the complexes administered. Intracellular conditions, such as i.e. pH, ionic concentrations and the presence of degrading enzymes are some of the limiting factors of intracellular stability of a compound. For example, if ester bonds are present in the complex they could be attacked by intracellular esterases. Further, unfavorable pH and ionic conditions may destabilize ionic linkages, e.g. when polycationic compounds such as poly-L-lysine are used to complex the DNA.
Another factor that may determine the success of a steroid mediated gene delivery approach is the maintenance of a high binding affinity between the steroid moiety and the steroid receptor after the derivatization of the steroid. The size of the DNA interacting moiety, the position of the linkage between the steroid and the DNA interacting moiety, as well as the steric properties of the linking bond itself, may, among others, determine whether steric hindrance of the steroid/steroid receptor interaction will occur after derivatization of the steroid. Further, complexing of the DNA to the DNA interacting moiety should be achieved at specific positions of the DNA molecule in order to avoid inactivation of the genes to be transcribed. For example, complexing the DNA by intercalation may randomly inactivate portions of the DNA. Further, the intercalating positions may even change after initial linkage was achieved, thereby preventing the use of two-step approaches that in a first step will link the DNA interacting moiety to a particular, i.e. non-transcribed DNA region and in a second step will ligate or clamp via bifunctional triple helix formers the functional genes to the complexed DNA region to avoid their inactivation. Also, complexation via cationic moieties interacting with the negatively charged DNA are random and may involve functionally important stretches of the DNA and thus interfere with the transcription of the complexed DNA.
A steroid mediated gene delivery system that combines high intracellular stability and a high binding affinity for the steroid receptor as well as the possibility for specific linkage to the desired DNA molecule has not been reported to date. Thus, there exists a continuous need for such a delivery system which is useful for the introduction of nucleic acids into the nuclei of cells, e.g., for the expression of therapeutical genes. The object of the present invention is therefore to provide such a novel system and new methods for introducing nucleic acids into the nuclei of cells, in particular mammalian cells.
The present invention provides for a compound comprising a steroid hormone linked to a DNA-interacting molecule. In a preferred embodiment, the steroid hormone is stably linked to the DNA-interacting molecule. This compound is useful for complexing with nucleic acids desired to be delivered to target cells.
In a further embodiment, the compound comprises a spacer between the steroid hormone and the DNA-interacting molecule.
This invention includes compounds, wherein the steroid hormone is selected from the group consisting of one or more of androgens, gestagens, oestrogens, glucocorticoids, mineralocorticoids, retinoids, thyroids or synthetic steroids.
Further included are compounds, wherein the DNA-interacting molecule is selected from the group consisting of one or more of intercalating agents, crosslinking reagents, incorporating molecules and ionically interacting molecules. In a preferred embodiment the DNA-interacting molecule is a psoralen.
In a further aspect the invention relates to a method for the preparation of the compound comprising the steps of ligating a steroid hormone to a DNA-interacting molecule. Such a method may involve the steps of ligating a spacer to the steroid hormone and ligating the DNA-interacting molecule to the spacer.
Furthermore, the present invention provides compounds that are complexed to a DNA molecule. In another aspect, this invention provides a method for the preparation of the complex comprising the steps of ligating a steroid hormone to a DNA-interacting molecule to form a compound and complexing the compound with a DNA molecule. This method may further comprise the steps of ligating a spacer to the steroid hormone and ligating the DNA-interacting molecule to the spacer.
In yet another aspect, the invention relates to the use of the compound for introducing a DNA molecule into the nucleus of a cell, in particular, into the nucleus of a non-dividing cell. A cell transfected with a complex of the invention as well as the use of such a cell for the medical treatment of a human being is also provided.
The invention also provides a pharmaceutical preparation comprising the complex of the invention and a physiologically tolerable carrier.
The invention further provides a method for transfecting cells comprising the step of administering a therapeutically effective amount of a complex of the invention to a subject.
It is still another object of the invention to provide an assay comprising the steps of a) transfecting cells with a complex of the invention, wherein the DNA molecule contains an expressible gene; b) monitoring the expression of said expressible gene, and c) comparing the expression of said expressible gene in transfected cells with the expression of said expressible gene in non-transfected cells.
The term xe2x80x9csteroid hormonexe2x80x9d as used for the purposes of the present invention includes other hormones or lipophilic ligands with unrelated structures and physiological purposes that function at the molecular level in a similar way to the steroid hormones. Thus, each molecule that is a small molecule that binds to a specific intracellular receptor that upon activation translocates to the nucleus of the cell is comprised in the meaning of a xe2x80x9csteroid hormonexe2x80x9d of the invention. Examples of such molecules other than androgens, gestagens, oestrogens, glucocorticoids, mineralocorticoids or synthetic steroids, in particular dexamethasone, are retinoids such as retinoic acid and 9-cis retinoic acid, thyroid hormones and vitamin D and their derivatives.
A steroid hormone of the invention is derivatized by stably linking it to a molecule that has the capability to interact with nucleic acids (referred to as xe2x80x9cDNA-interacting moleculexe2x80x9d). The derivatized steroid then may be complexed with a nucleic acid via its DNA-interacting moiety (xe2x80x9cnucleic acid/compound complexxe2x80x9d, herein also referred to as the xe2x80x9ccomplexxe2x80x9d of the invention). The DNA molecule may be complexed to one or more compounds via intercalation, crosslinking, incorporation, ionical or hydrophobical interaction. Thus xe2x80x9cto complexxe2x80x9d according to this invention includes linking the nucleic acid by ionical, hydrophobic and covalent interaction, and, accordingly, a complex of the invention includes all molecules wherein a compound of the invention is linked to a nucleic acid, independent of the chemical type of linkage/bond formation.
The nucleic acid/steroid complex of the invention can be transfected into cells and bind to the cytosolic steroid hormone receptors, which subsequently mediate nuclear localization of the complex. The nuclear localization of transfected DNA will enable the expression of genes encoded by the nucleic acids. The present invention thereby provides an improved method for delivering nucleic acids to the nuclei of cells, in particular, mammalian cells, e.g. exogenous DNA for transforming human cells. This improved method for example generally comprises providing to the cell targeted for transformation a specifically designed nucleic acid/compound complex, comprising the exogenous DNA desired to be targeted to the nucleus and expressed in the transformant.
The present invention provides compounds to form complexes with nucleic acids and the nucleic acid/compound complexes themselves. The present invention further provides an improved method for transforming cells with exogenous nucleic acids such as e.g. DNA, using such nucleic acid/compound complexes. This method combines positive attributes of viral (cell type specificity, nuclear targeting) and non-viral (convenience of preparation and application, safety, less limitations as to DNA size) methods of transfection and subsequent transformation.
According to the invention, the nucleic acid/compound complex comprising the exogenous nucleic acid, such as e.g. an steroid hormone linked to a DNA sequence encoding a therapeutic gene, may be delivered to the cell by means such as, but not restricted to, electroporation, microinjection, induced uptake, microprojectile bombardment, liposomes, viral vectors or other means as are known in the art. Accordingly, the present invention provides novel means for the in vivo and ex vivo/in vitro transformation and integration of exogenous nucleic acids desired to be expressed within hosts or host cells, particularly for the purpose of gene therapy. In one embodiment the 11xcex2-hydroxysteroid dehydrogenase (11xcex2-OHSD) gene is transfected into and expressed in 11xcex2-OHSD deficient cells for the treatment of Apparent Mineralocorticoid Excess (AME). AME is characterized by an impaired conversion of cortisol by the enzyme 11xcex2-hydroxysteroid deghydrogenase and is associated with a severe low renin, low aldosterone and hypertension with hypokalemia.
In the course of the experiments which led to the present invention it has been found that cells can efficiently be transformed by using a nucleic acid/compound complex of the invention.
The DNA-interacting molecule of the compound may be selected from the group consisting of one or more of intercalating agents, crosslinking reagents, incorporating molecules, tonically or hydrophobically interacting molecules. Preferred is a compound wherein the DNA-interacting molecule is psoralen (aminotrioxsalen). Psoralen is a molecule that can be specifically crosslinked to parts of a DNA molecule after photoactivation. Preferred ionically interacting molecules of the invention are polycations, in particular spermidine, spermine, polylysine and protamine.
A steroid hormone according to the invention may be xe2x80x9clinkedxe2x80x9d to a DNA-interacting molecule directly via a covalent, ionic or hydrophobic interaction. In the alternative, it may be indirectly linked to a DNA-interacting molecule with a xe2x80x9cspacerxe2x80x9d being positioned between the steroid hormone and the DNA-interacting molecule. Compounds including a spacer between the steroid hormone and the DNA-interacting molecule are preferred. A xe2x80x9cspacerxe2x80x9d of the invention may for example be selected from the group of dicarbonic acids, i.e. succinates, in particular a hemisuccinate, ether, and thioether, amino acids, amines etc. In a preferred embodiment urethanes are spacers used in this invention. The compound may preferably comprise a spacer between the steroid hormone and the DNA-interacting molecule comprising more than two atoms. More preferred is a spacer comprising 2-30 atoms, particularly preferred is a spacer having 5-15 atoms. Most preferred is a spacer having 9 to 11, and in particular 10 atoms. Preferred are spacers wherein the atoms are C, O, N and S; spacers containing xe2x80x94Sxe2x80x94Sxe2x80x94 or xe2x80x94Oxe2x80x94Oxe2x80x94 are excluded from this invention. Within the meaning of the present invention, the spacer comprises the atoms between the first carbon atom which is not derived from the steroid hormone, as well as the first carbon atom derived from the DNA-interacting molecule, and it further includes said first atoms not derived from either the steroid hormone or the DNA-interacting molecule. For example, within the meaning of this invention, the spacer of Formula 29 which links Dexamethasone and Spermidine has a length of 10 atoms.
In a preferred embodiment of this invention the link is stable within the intracellular environment to which it exposed after cellular uptake. In another preferred embodiment of this invention the link is stable within blood serum or plasma. In particular, the link is stable under acidic (pH 5) and alkaline (pH 9) conditions, by proteinase K (pH 7.8, 50 xcexcg/ml) and dispase (2.4 U/ml) digestion and after incubation with cellular extracts and with DMEM/10% FCS.
The bond linking the steroid to the spacer or the DNA-interacting molecule may be positioned depending on their chemical accessibility and on their influence on the affinity for the cognate receptor. Preferred are positions either at carbon atom 1, 2, 4, 6, 7, 11xcex1, 12, 15, 16, 17 or 21 if the steroid hormone is a glucocorticoid. If the steroid hormone is an androgen, positions 1, 2, 4, 6, 7, 11xcex1, 12, 15, 16, 17 are preferred. Preferred is an urethane bond positioned either at carbon atom 6 or 21 of a glucocorticoid. More preferred is an urethane bond positioned at carbon atom 21 of a glucocorticoid.
The present invention thus provides for a compound comprising a steroid hormone linked to a DNA-interacting molecule. In a preferred embodiment, it is stably linked to the DNA-interacting molecule via a covalent bond, e.g. a urethane bond, a thiourethane bond, a sulfonate, or an ether bond. These covalent bonds provide for high intracellular stability of the compound as well as serum/plasma stability. In a preferred embodiment the bond is a urethane bond. In a more preferred embodiment the steroid hormone is linked via a first urethane bond to a spacer and the spacer is linked via a second urethane bond to the DNA-interacting molecule.
The present invention further provides a method for the preparation of a complex comprising the steps of ligating a steroid hormone to a DNA-interacting molecule to form a compound and complexing the compound with a DNA molecule. The method may further comprise the steps of ligating a spacer to the steroid hormone and ligating the DNA-interacting molecule to the spacer. Most preferred is the method wherein the steroid hormone is linked via an urethane bond to the DNA-interacting molecule. Depending on the DNA-interacting molecule used, the DNA molecule may be complexed to one or more compounds via intercalation, crosslinking, incorporation, ionical or hydrophobical interaction. In a preferred embodiment the DNA molecule is crosslinked to the compounds. In a preferred embodiment the crosslinking reagent is psoralen. To achieve xe2x80x9cincorporationxe2x80x9d the steroid is conjugated via a suitable spacer to a desoxy-ribonucleotide triphosphate which is then build into a DNA molecule by a polymerase-mediated protocol, i.e. nick-translation, 5xe2x80x2 overhangs filling or PCR incorporation.
In one embodiment this invention relates to the use of the compound for introducing a DNA molecule into the nucleus of a cell, in particular a non-dividing cell, and in a particular embodiment, a quiesent somatic cell.
A method for transfecting cells comprising the step of administering a therapeutically effective amount of a nucleic acid/compound complex to a subject, in particular a human being, is also provided by the present invention. In an embodiment the 11xcex2-hydroxysteroid deghydrogenase (11xcex2-OHSD) gene is administered in a therapeutically effective amount in 11xcex2-OHSD deficient cells for the treatment of Apparent Mineralocorticoid Excess (AME). This invention includes, but is not limited to the delivery of, for example, mammal-specific genes, such as the insulin gene, the somatostatin gene, the interleukin genes, the t-PA gene, etc. Apart from naturally occurring structural genes that code for a useful and desirable property or a pharmacological agent, within the scope of this invention it is also possible to use genes that have been modified previously in a specific manner using chemical or genetic engineering methods.
The term xe2x80x9cDNAxe2x80x9d or xe2x80x9cnucleic acidxe2x80x9d as a component of the xe2x80x9cnucleic acid/steroid complexxe2x80x9d according to the present invention may be any type of nucleic acid, for example RNA, modified RNA or DNA, wherein DNA is the preferred form. For example, the present invention particularly provides an improved method for transiently transfecting and for stably transforming cells with exogenous nucleic acids such as e.g. the 11xcex2-hydroxysteroid deghydrogenase gene. The term xe2x80x9cexogenousxe2x80x9d DNA or nucleic acid used herein is meant to include any DNA or other nucleic acid that has been obtained by recombinant nucleic acid technology. The exogenous DNA to be used in the process according to the invention for transforming cells may be either of homologous or heterologous origin with respect to the cell type involved or it may be of synthetic origin or both. The coding DNA sequence can be constructed according to conventional methods, e.g. from genomic DNA, or from cDNA. Another possibility is the construction of a hybrid DNA sequence consisting of both cDNA and genomic DNA and/or synthetic DNA. The cDNA may originate from the same gene as the genomic DNA, or alternatively both the cDNA and the genomic DNA may riginate from different genes. In any case, however, both the genomic DNA and/or the cDNA may each be prepared individually from the same or from different genes. The term DNA or nucleic acid includes (a) DNA sequences that have been been prepared entirely or at least partially by chemical means and (b) antisense or sense oligonucleotides. For example, synthetic DNA sequences may be suitably used, e.g. for modifying native DNA sequences in terms of codon usage, expression efficiency, etc. If the DNA sequence to be transformed into the recipient animal cell contains portions of more than one gene, these genes may originate from one and the same organism, from several organisms that belong to more than one strain, one variety or one species of the same genus, or from organisms that belong to more than one genus of the same or of another taxonomic unit.
In a particular embodiment of this invention the DNA complexed to the compound of the invention may be used as a link to another DNA that contains i.e. a therapeutic gene. For example, the DNA that contains i.e. a therapeutic gene-may be directly ligated or clamped i.e. via bifunctional triple helix formers to the stretch of DNA that has been complexed to the compound of the invention.
Chimeric recombinant DNA molecules that comprise an expressible DNA, but especially a structural gene, preferably a heterologous structural gene operably linked with expression signals active in animal cells, such as enhancer, promoter and transcription termination sequences, as well as, optionally, with further coding and/or noncoding sequences of the 5xe2x80x2 and/or 3xe2x80x2 region such as e.g. signal sequence may also be preferably used within the transformation process as part of the nucleic acid/compound complex used according to the present invention. It is often advantageous to incorporate a leader sequence between the promoter sequence and the adjacent coding DNA sequence, the length of the leader sequence being so selected that the distance between the promoter and the DNA sequence to be expressed is the optimum distance for expression of the associated structural gene.
The expression signals active in mammalian cells usually comprise a promoter that is recognised by the host organism and is operably linked to the DNA to be expressed in the transformant. Such a promoter may be inducible or constitutive. The promoters are operably linked to said DNA by removing the promoter from the source DNA by restriction enzyme digestion and combining the isolated promoter sequence with the expressible DNA sequence. Both the native promoter sequence of the structural gene of interest and many heterologous promoters may be used to direct amplification and/or expressionl of said structural gene. Suitable promoters for animal and in particular mammalian hosts are those derived from the genomes of viruses such as polyoma virus, adenovirus, fowlpox virus, bovine papilloma virus, avian sarcoma virus, Rouse sarcoma virus (RSV), cytomegalovirus (CMV), a retrovirus and Simian Virus 40 (SV40), from heterologous mammalian promoters such as the actin promoter or a very strong promoter, e.g. a ribosomal protein promoter, and from the promoter normally associated with structural gene sequence to be expressed, provided such promoters are compatible with the host cell systems.
The transcription of an exogenous DNA encoding the desired structural gene can be increased by inserting an enhancer sequence into the DNA as a component of the nucleic acid/compound complex according to the invention. Enhancers are relatively orientation and position independent. Many enhancer sequences are known from mammalian genes (e.g. elastase and globin). However, typically one will employ an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270) and the CMV early promoter enhancer. The enhancer may be spliced into the recombinant chimeric sequence at a position 5xe2x80x2 or 3xe2x80x2 to the coding DNA sequence, but is preferably located at a site 5xe2x80x2 from the promoter.
Host cells to which nucleic acids can be delivered by a method according to the invention include insect and vertebrate cells. In recent years propagation of vertebrate ceils in culture (tissue culture) has become a routine procedure. Examples of useful vertebrate host cell lines are epithelial or fibroblastic cell lines such as Chinese hamster ovary (CHO) cells, COS1 cells (monkey kidney cells transformed with SV40 T-antigen), CV1 cells (parent line of the former), Rat1 (rat fibroblast) cells, NIH 3T3 cells, HeLa cells, LLC-Pk1 (pig kidney epithelial) cells or 293T cells. The host cells referred to in this disclosure comprise cells in in vitro/ex vivo culture as well as cells that are within a host animal.
Especially suitable for use in the process according to the invention are all those structural genes which upon expression produce proteins or polypeptides which are beneficial for the transformed cells, tissues or mammals, e.g. which compensate eventual mutatations, or which have pharmacological properties and could be used as pharmaceutical agents in the treatment of diseases. Examples for such structural genes include those encoding hormones, immunomodulators and other physiologically active substances.
Furthermore, the broad concept of the present invention also includes genes that are produced entirely or partially by chemical synthesis. Genes or DNA sequences that may be used within the scope of the present invention are therefore both homologous and heterologous gene(s) or DNA and also synthetic gene(s) or DNA according to the definition given within the scope of the present invention.
Alternatively, oligonucleotides can be used corresponding in sequence to a cellular sequence to be targeted, either in the same coding direction, as such or carrying a mutation, or in the antisense coding direction.
Possible methods for the direct transfer of the nucleic acid/compound complex according to the invention into a cell comprise, for example, the treatment of cells using procedures that modify the plasma membrane, for example, polyethylene glycol treatment, liposome-based technologies, heat shock treatment or electroporation, or a combination of those procedures (see e.g. Chu et al. (1987); Hodgson and Solaiman (1996), Shillito et al. (1985)).
In the electroporation technique animal cells together with the nucleic acid/compound complex used according to the invention are subjected to electrical pulses of high field strength. This results in a reversible increase in the permeability of biomembranes and thus allows the insertion of the nucleic acid/compound complex according to the invention. Electroporated cells renew their cell membrane, divide and form aggregates or monolayers of transformed cells. Selection of the transformed cells can take place with the aid of the above-described phenotypic markers.
Also suitable for the transformation of mammalian cells is direct gene transfer using co-transformation (Schocher R J et al, (1986)). Co-transformation is a method that is based on the simultaneous taking up and integration of various DNA molecules (non-selectable and selectable genes) into the genome and that therefore allows the detection of cells that have been transformed with non-selectable genes.
Further, means for inserting the nucleic acid/compound complex used according to the invention directly into a cell comprise using purely physical procedures, for example by microinjection using finely drawn micropipettes or by bombarding the cells with microprojectiles that are coated with the transforming or transiently transfecting nucleic acid (Wang Y-C et al, (1988)) or are accelerated through a nucleic acid containing solution in the direction of the cells to be transformed by a pressure impact thereby being finely atomized into a fog with the solution as a result of the pressure impact (EP-A434,616). Microprojectile bombardment has been advanced as an effective transformation technique for animal cells.
The list of possible transformation and transfection methods given above by way of example is not claimed to be complete and is not intended to limit the subject of the invention in any way.
The method according to the invention can be advantageously used to increase the transformation efficiency of transformation processes, in that, for example, less transforming DNA is needed as compared to the conventional techniques. Additionally, the present invention can be used for somatic gene therapy in humans, which use is also part of the invention.
In various alternative embodiments of the present invention, therapeutic compositions useful for practicing the therapeutic methods described herein are contemplated. As used herein, the terms xe2x80x9ctherapeutic compositionsxe2x80x9d and xe2x80x9cpharmaceutical preparationsxe2x80x9d are used interchangeably. Therapeutic compositions of the present invention may contain a physiologically tolerable carrier together with one or more therapeutic nucleic acid/compound complexes of this invention, dissolved or dispersed therein as an active ingredient. The nucleic acid/compound complexes in the therapeutic compositions may have been combined with/introduced into a transfecting agent. A xe2x80x9ctransfecting agentxe2x80x9d in the sense of this invention may be any agent presently known or unknown that improves transfection of mammalian cells when administered as part of or together with a DNA to be transfected. Thus, the present invention comprises therapeutic compositions useful in the specific targeting of as well as in delivering a therapeutic nucleotide sequence to those cells. In a preferred embodiment, the therapeutic composition is not immunogenic or otherwise able to cause undesirable side effects when administered to a subject for therapeutic purposes.
As used herein, the terms xe2x80x9cpharmaceutically acceptablexe2x80x9d, xe2x80x9cphysiologically tolerablexe2x80x9d and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a subject, e.g., a mammal, without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art. Typically such compositions are prepared as injectables, either as liquid solutions or suspensions. However, solid forms suitable for solution or suspension in liquid prior to use can also be prepared. A preparation can also be emulsified, or formulated into suppositories, ointments, creams, dermal patches, or the like, depending on the desired route of administration.
The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof, including vegetable oils, propylene glycol, polyethylene glycol and benzyl alcohol (for injection or liquid preparations); and petrolatum (e.g., VASELINE), vegetable oil, animal fat and polyethylene glycol (for externally applicable preparations). In addition, if desired, the composition can contain wetting or emulsifying agents, isotonic agents, dissolution promoting agents, stabilizers, colorants, antiseptic agents, soothing agents and the like additives (as usual auxiliary additives to pharmaceutical preparations), pH buffering agents and the like which enhance the effectiveness of the active ingredient.
The therapeutic compositions of the present invention can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.
Physiologically tolerable carriers are well known in the art. Exemplary of liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
A therapeutic composition typically contains an amount of a nucleic acid/steroid complex of the present invention sufficient to deliver a therapeutically effective amount to the target tissue, typically an amount of at least 0.01 weight percent to about 1 weight percent of therapeutic nucleolide sequence per weight of total therapeutic composition. A weight percent is a ratio by weight of therapeutic nucleotide sequence to total composition. Thus, for example, 0.01 weight percent is 0.01 grams of DNA segment per 100 grams of total composition. However, lower concentrations i.e. 0,0001% can be used if DNA transfer reaches efficiencies comparable to viral transfer, higher concentrations i.e. up to 10% can be reached if special formulations allowing higher DNA solubility can be developed.
The nucleic acid/compound complexes of the present invention are particularly suited for gene therapy. Thus, various therapeutic methods are contemplated by the present invention. Methods of gene therapy are well known in the art (see, e.g., Larrick and Burck (1991); Kriegler (1990)). The term xe2x80x9csubjectxe2x80x9d should be understood to include any animal, particularly mammalian, patient, such as any murine, rat, bovine, porcine, canine, feline, equine, ursine, or human patient.
When the foreign gene carried encodes a tumor suppressor gene or another anti-tumor protein, the compounds of the present invention are useful to treat or reduce hyperproliferative cells in a subject. Hyperproliferation and/or uncontrolled proliferation occurs first, in neoplastic diseases, second, in so-called immune mediated entities and third, in disease states with hyperplasia/hypertrophy. Examples of the first group are neoplasias such as breast cancer, bladder cancer, colon cancer, lung cancer, various leukemias, lymphomas, sarcomas and others. In particular, tumours that may produce steroid hormone receptors, such as colon cancer, rectal cancer, breast cancer, prostate cancer, endometrial cancer, ovarian cystadenocarcinoma, pancreatic cancer, lung cancer, in particular non-small cell lung cancer, gallbladder cancer and thyroid carcinoma are contemplated. To the second group belong among others rejection of transplanted organs, systemic immune mediated diseases such a lupus erythematodes, periartheritis nodosa, Wegeners disease, asthma, eczema etc. The third entity comprises etiologically often poorly understood local or diffuse organomegaly, such as benign prostatic hypertrophy, psoriasis, hypertrophic cardiomyopathy, thyroid hyperplasia etc.
In all these disease states the present approach will be of great interest whenever a particular ligand receptor is restrictively expressed in this target cell population.
Administration includes, but is not limited to, the introduction of therapeutic compounds (=compositions and complexes of the present invention) into a cell or subject via various means, including direct injection, intravenously, intraperitoneally, via intra-tumor injection, via aerosols, or topical administration, as disclosed herein may also be combined for administration of an effective amount of the compounds with a pharmaceutically-acceptable carrier, as described herein.
As used herein, xe2x80x9ceffective amountxe2x80x9d of a therapeutic compound generally means the amount of therapeutic composition (or nucleic acid/steroid complexes or protein expression produced thereby) which achieves a positive outcome in the subject to whom the therapeutic compound is administered. The total volume administered will necessarily vary depending on the mode of administration, as those of skill in the relevant art will appreciate, and dosages may vary as well.
The present invention also contemplates methods of ameliorating pathologies characterized by genetic defects in a subject, by administering to the subject an effective amount of a therapeutic compound as described herein. The nucleic acid portion of such a therapeutic compound preferably contains a foreign gene encoding a gene product (e.g. polypeptide or protein) having the ability to ameliorate the pathology, under suitable conditions. As used herein, the term xe2x80x9cgenetic defectxe2x80x9d means any disease, condition or abnormality which results from inherited and/or aquired changes causing directly disease states and/or predisposing to aquired and/or degenerative disease states. Groups of diseases comprise among others, disorders ot the carbohydrate metabolism, inborn errors of amino acid-, organic acid-, purine or pyrimidine-metabolism, lysosomal storage diseases, peroxysomal disorders, cystic fibrosis, sickle cell disease, AME or degenerative diseases presently poorly understood.
For in vitro gene transfer, administration is often accomplished by first isolating a selected cell population from a patient such as lung epithelial cells, lymphocytes and the like followed by in vitro gene transfer of the complex of this invention and the replacement of the cells into the patient. In vivo therapy is also contemplated, e.g., via the administration of therapeutic compositions of this invention by various delivery means. For example, aerosol administration and administration via subcutaneous, intravenous, intraperitoneal, intramuscular, ocular means and the like are also within the scope of the present invention. Other gene-delivery methods are also useful in conjunction with the methods, compositions and constructs of the present invention; see, e.g., published International Application No. WO 95/11984, the disclosures of which are incorporated by reference herein.
The present invention also contemplates various methods of targeting specific cells, e.g. cells in a subject in need of diagnosis and/or treatment. As discussed herein, the present invention contemplates that the compositions of the present invention may be directed to specific receptors or cells, in particular, steroid receptors and steroid receptor expressing cells, for the ultimate purpose of delivering DNA to the nuclei of specific cells or cell types. The compounds of the present invention are particularly useful in this regard. A special feature of the complexes used according to the invention is their ability to also target non dividing cells, due to their nuclear targeting potential.